[16] Studies of base pair kinetics by NMR measurement of proton exchange

This chapter discusses the studies of base pair kinetics by nuclear magnetic resonance (NMR) measurement of proton exchange. Base pairing is ubiquitous in nucleic acids; it plays a key role in the structure and function of biological molecules (B-DNA, tRNA, etc.) and of biochemical constructs in PCR, antisense strategy, etc. The systematic disruption of base pairs is a prerequisite for replication and transcription of double-stranded DNA. Local disruption could play a role in mechanical properties of nucleic acids and in the specificity of protein-nucleic acid recognition. It is involved in chemical reactions of nucleic acids. As a probe of structural properties, the kinetics of base pair opening forms a useful complement to structure determination by NMR. The most common approach to base pair opening is the measurement of imino-proton exchange, with water, by proton NMR. With NMR, an exchange event can be directly assigned to an imino (rather than amino or ribose) proton of a specific base pair, in contrast to the earlier procedures based on isotope exchange, monitored by radioactivity or ultraviolet, infrared, or Raman spectroscopy. The measurement methods are of two types: the kinetics of real-time exchange and of magnetization transfer measure exchange times directly, whereas the measurements of longitudinal relaxation and line broadening can only determine the differential effect of added catalyst on the exchange time.